The telomere and centromere are two specialized structures of eukaryotic chromosomes that are essential for chromosome stability and segregation. These structures are usually characterized by large tracts of tandemly repeated DNA. In mouse, the two structures are often located in close proximity to form telocentric chromosomes. To date, no detailed sequence information is available across the mouse telocentric regions. The antagonistic mechanisms for the stable maintenance of the mouse telocentric karyotype and the occurrence of whole-arm Robertsonian translocations remain enigmatic. We have identified large-insert fosmid clones that span the telomere and centromere of several mouse chromosome ends. Sequence analysis shows that the distance between the telomeric T 2AG3 and centromeric minor satellite repeats range from 1.8 to 11 kb. The telocentric regions of different mouse chromosomes comprise a contiguous linear order of T2AG3 repeats, a highly conserved truncated long interspersed nucleotide element 1 repeat, and varying amounts of a recently discovered telocentric tandem repeat that shares considerable identity with, and is inverted relative to, the centromeric minor satellite DNA. The telocentric domain as a whole exhibits the same polarity and a high sequence identity of >99% between nonhomologous chromosomes. This organization reflects a mechanism of frequent recombinational exchange between nonhomologous chromosomes that should promote the stable evolutionary maintenance of a telocentric karyotype. It also provides a possible mechanism for occasional inverted mispairing and recombination between the oppositely oriented TLC and minor satellite repeats to result in Robertsonian translocations.centromere ͉ chromosome ͉ evolution ͉ telomere

Neocentromeres (NCs) are fully functional centromeres that arise ectopically in noncentromeric regions lacking ␣-satellite DNA. Using telomere-associated chromosome truncation, we have produced a series of minichromosomes (MiCs) from a mardel(10) marker chromosome containing a previously characterized human NC. These MiCs range in size from Ϸ0.7 to 1.8 Mb and contain single-copy intact genomic DNA from the 10q25 region. Two of these NC-based Mi-Cs (NC-MiCs) appear circular whereas one is linear. All demonstrate stability in both structure and mitotic transmission in the absence of drug selection. Presence of a functional NC is shown by binding a host of key centromereassociated proteins. These NC-MiCs provide direct evidence for mitotic segregation function of the NC DNA and represent examples of stable mammalian MiCs lacking centromeric repeats. M ammalian artificial chromosomes have several potential biotechnological and therapeutic applications arising from their ability to exist episomally, carry large DNA inserts, and allow expression of genes independently of the host genome. By analogy with their yeast counterparts, it has been assumed that mammalian artificial chromosomes require a functional mammalian centromere, telomeres, and DNA replication origins for proper segregation. At present, the least understood and most complex of these three components is the centromere.The identification of many protein components necessary for correct centromere activity, and the characterization of centromere DNA sequences in a variety of species, have greatly increased our knowledge of the mechanisms underlying centromere formation and function (1-3). This knowledge has facilitated the development of a number of strategies for mammalian artificial chromosome construction. One strategy involves the de novo formation of artificial chromosomes by transfection of large arrays of human ␣-satellite into human cells (4-7). Although some of the generated artificial chromosomes were linear in structure (4), others were consistently circular (5,7,8) and all were typically 1 or more orders of magnitude larger than the input DNA.A second strategy involves the use of telomere-associated chromosome truncation to remove nonessential chromosome arms to produce minichromosomes (MiCs) in situ. Sequential truncation of a human X and Y chromosome has yielded a number of ␣-satellitecontaining MiCs of varying stability and sizes ranging from Ϸ0.7 Mb to more than 4 Mb (9-15). A final strategy for production of mammalian artificial chromosomes involves the amplification of pericentric DNA followed by controlled breakage of chromosomes to produce satellite DNA-based artificial chromosomes of between 60 and 400 Mb (16,17). These artificial chromosomes express exogenous genes and can be stably introduced into different mammalian cell lines and transgenic mice (18)(19)(20).Neocentromeres (NCs) lacking the repeat sequences traditionally associated with centromere function recently have been described (21,22). Characterization of NCs in humans sugg...